Throughout this application various publications are referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
1. Field of the Invention
The present invention is related to the medical arts, particularly to the field of gene therapy.
2. Discussion of the Related Art
Viruses have been tested for their ability to treat various types of malignancies in animals and humans. The proposed therapeutic mechanisms of viral tumor therapy in the prior art include: (i) producing new antigens on the tumor cell surface to induce immunologic rejection, called xe2x80x9cxenogenization,xe2x80x9d and (ii) direct killing of the tumor cell by a virus, called xe2x80x9concolysis.xe2x80x9d
Several animal models and animal types of malignant tumor have been used to study oncolysis with wild-type viruses. (Moore, Ann. Rev. Microbiol. 8: 393 [1954]; Moore, Progr. Exp. Tumor Res. 1:411 [1960]). At least nine viruses have been shown to be capable of inducing some degree of tumor regression of a variety of tumors in animals. A major drawback found in these early studies, however, was systemic infection of the patient by the virus.
Later in the quest for a viral therapy for cancer, clinical trials employing herpes viral vector therapy were approved in the United States to treat human tumors. (Culver, Clin. Chem. 40: 510 [1994]). These studies employed replication-incompetent or defective viruses to potentially overcome the problem of systemic infection by the virus. However, the use of replication-defective herpes viruses for treating malignant tumors requires producer cells because each replication-defective herpes virus particle can enter only a single cell and cannot productively infect others thereafter. Thus, due to their inability to replicate, replication-defective herpes viruses cannot spread to other tumor cells, they are unable to penetrate a deep, multilayered tumor in vivo. (Markert et al., Neurosurg. 77: 590 [1992]).
On the other hand, the herpes simplex virus type 1 (HSV-1) appears to be particularly well suited for use in the treatment of malignancies. Mutation of several of the viral genes involved in DNA replication, including UTPase and thymidine kinase- render the virus replication-defective in normal postmitotic cells, like neurons, but replication-competent in dividing cells, which can complement the defect. In addition, the HSV-thymidine kinase (TK) gene product can convert the anti-herpes drugs gancyclovir or acyclovir to nucleotide analogs which block both viral and cellular replication, thereby killing dividing tumor cells.
The need for a safe and effective HSV-1-derived vector is especially acute with respect to malignant tumors of the central nervous system. These malignancies are usually fatal, despite recent advances in the areas of neurosurgical techniques, chemotherapy and radiotherapy. In particular, there are no standard therapeutic modalities that can substantially alter the prognosis for patients with malignant tumors of the brain, cranium, and spinal cord. For example, high mortality rates persist for patients diagnosed with malignant medulloblastomas, malignant meningiomas, malignant neurofibrosarcomas and malignant gliomas, which are characterized by infiltrative tumor cells throughout the brain. Although intracranial tumor masses can be debulked surgically, treated with palliative radiation therapy and chemotherapy, the survival associated with a diagnosis of glioma, especially glioblastoma, is typically measured in months.
Oldfield et al. introduced viral vectors carrying the herpes simplex virus (HSV)-1 thymidine kinase (HS-tk) gene into brain tumor cells in human patients. (Oldfield et al., Human Gene Therapy 4: 39 [1993]). In this study, there was some evidence of anti-tumor effect in five of the eight patients in the clinical trial. However, none of the patients was cured of brain cancer. Some of the limitations of current viral based therapies, described by Oldfield, include: (1) the low titer of virus produced; (2) virus spread limited to the region surrounding the producer cell implant; (3) possible immune response to the producer cell line; (4) possible insertional mutagenesis and transformation of virally infected cells; (5) a single treatment regimen of the drug, gancyclovir, because the xe2x80x9csuicidexe2x80x9d product kills virally infected cells and producer cells; and (6) the bystander effect of killing being limited to cells in direct contact with the virally transformed cells. (Bi, W. L. et al., Human Gene Therapy 4: 725 [1993]).
During the early 1990""s, the use of genetically engineered replication-competent HSV-1 viral vectors was first explored in the context of finding an antitumor viral therapy. Replication-competent mutants of herpes simplex virus type I (HSV-1) with single mutations demonstrated therapeutic potential against experimental malignant brain tumors, while being attenuated for neurovirulence. It was thought that a replication-competent virus would have the advantage of being able to enter one tumor cell, make multiple copies of its genome, lyse the cell and spread to other tumor cells. A thymidine kinase-deficient (TKxe2x88x92) mutant, dlsptk, was able to destroy human malignant glioma cells implanted into the brain of an animal. (Martuza et al., Science 252: 854 [1991]). The major disadvantage to this system was that these TKxe2x88x92 mutants were only moderately attentuated for neurovirulence, i.e., the ability to replicate in brain cells causing inflammation of the brain, and they produced encephalitis at the doses required to kill the tumor cells adequately. (J. M. Markert et al., Neurosurgery 32: 597 [1993]). Roizman described a HSV-based Txe2x88x92 vector system capable of expressing foreign genes inserted into the TK gene. (Roizman, Herpes Simplex Virus as a Vector, U.S. Pat. Nos. 5,599,691 and 5,288,641). Residual neurovirulence of TKxe2x88x92 limits the usefulness of such vectors for tumor therapy.
Other single mutants of HSV-1 included hrR3, containing an insertion of the Escherichia coli lacZ gene into the viral ICP6 gene, which encodes the ribonucleotide reductase large subunit (T. Mineta et al., Gene Therapy 1:S78 [1994]; T. Mineta et al., J. Neurosurg. 80:381 [1994]) and R3613 containing deletions in both copies of the xcex334.5 gene, a neurovirulence gene. (Markert et al., Neurosurgery 32:597 [1993]). Roizman described a recombinant, purportedly avirulent HSV lacking the ability to express a functional xcex334.5 gene product, a neurovirulence factor. (Roizman, Recombinant Herpes Simplex Viruses vaccines and methods, U.S. Pat. No. 5,328,688). Spontaneous reactivation rates of these mutants was only relatively attenuated, not entirely eliminated. (E.g., G.-C. Perng et al, J. Virol. 70(5):2883-93 [1996]; G.-C. Perng et al., J. Virol. 69(5):3033-411 [1995]).
Multi-mutated HSV-1 mutants have been described having augmented safety. Multiple mutations engineered into HSV-1 made the possibility of reversions of wild type unlikely and confirmed multiple and potentially synergistic safety features by attenuating of multiple mechanisms of virulence. Kramm et al. reported a HSV-1 mutant vector, MGH-1, defective for both ribonucleotide reductase and xcex334.5, which had higher therapeutic safety than hrR3, but had clearly decreased therapeutic efficiency compared to hrR3. (C. M. Kramm et al., Therapeutic efficiency and safety of a second-generation replication-conditional HSV1 vector for brain tumor gene therapy, Hum. Gene Ther. 8(17):2057-68 [1997]). Martuza et al. taught a replication competent HSV-1 vector having deletions in both of its xcex334.5 genes, as well as in the ICP6 gene which encodes for the large sub unit of the HSV ribonucleotide reductase. (Martuza et al., Replication-competent Herpes Simplex Virus mediates destruction of neoplastic cells, U.S. Pat. No. 5,585,096). Martuza et al., taught a HSV-1-derived replication competent vector for tumor therapy that was driven by a tumor-specific or cell-specific promoter and was purportedly not neurovirulent. (Martuza et al., Tumor- or cell-specific Herpes Simplex Virus replication, U.S. Pat. No. 5,728,379). Although improved in safety, these mutants have shown attenuated therapeutic potential.
Even while some of these mutants, for example, xcex334.5 mutants, have been shown to reduce neurovirulence by as much as 100,000 fold in mice, a major problem with existing technology is the spontaneous reactivation of HSV-1. Following infection at a peripheral site, HSV-1 establishes a life long latent infection of the sensory neurons enervating the peripheral site. In the absence of viral reactivation, latency is totally benign, with no known pathology. In rabbits, which can have rates of spontaneous HSV-1 reactivation comparable to those seen in humans, HSV-1 xcex334.5 mutants have greatly reduced spontaneous reactivation when rabbits are infected at xe2x80x9cnormalxe2x80x9d doses (2xc3x97105 pfu/eye). (G.-C. Perng et al., An Avirulent ICP34.5 deletion mutant of Herpes Simplex Virus Type 1 is capable of in vivo spontaneous reactivation, J. Virol. 69(5):3033-41 [1995]). In contrast, at extremely high infectious doses (2xc3x97108 pfu/eye) wild type spontaneous reactivation rates are achieved, separably from level of neurovirulence. (G. C. Perng et al., High-dose ocular infection with a Herpes Simplex Virus Type 1 ICP34.5 deletion mutant produces no corneal disease or neurovirulence yet results in wild-type levels of spontaneous reactivation, J. Virol. 70(5):2883-93 [1996]).
During neuronal latency, latency-associated transcript (LAT) is the only viral gene that is abundantly transcribed. This is a function of the LAT promoter, a very powerful promoter with significant neuronal specificity. LAT is essential for efficient spontaneous reactivation. In rabbits, HSV-1 LAT null mutants have spontaneous reactivation rates of ⅓ or less than that of wild type or rescued viruses. (G. C. Perng et al., The Latency-Associated Transcript gene of Herpes Simplex Virus Type 1 (HSV-1) is required for efficient in vivo spontaneous reactivation of HSV-1 from latency, J. Virol. 68(12):8045-55 [1994]). But this is still a substantial level of spontaneous reactivation.
Therefore, there is a desideratum for an effective HSV vector for brain tumor therapy that is non-neurovirulent and also does not reactivate spontaneously.
Delivery of the virus particles to tumor cells is another problem encountered in HSV therapy against brain tumors. Stereotactic inoculation of virus particles directly into a brain tumor has commonly been limited to small tumors, because the small volume of distribution by stereotactic inoculation limits the efficacy of viral therapy for large and disseminated tumors. (L. L. Muldoon et al., Comparison of intracerebral inoculation and osmotic blood-brain disruption for delivery of adenovirus, herpesvirus, and iron oxide particles to normal rat brain, Am. J. Pathol. 147(6):1840-51 [1995]).
Kramm et al. described intrathecal injection of HSV vector hrR3 into the cerebrospinal fluid of rats with gliosarcomas, which resulted in HSV expression in frontal tumors and leptomeningeal tumor foci along the entire neuroaxis; however there was substantial toxicity associated with intrathecal injection of the vector. (C. M. Kramm et al., Herpes vector-mediated delivery of marker genes to disseminated central nervous system tumors, Hum. Gene Ther. 7(3):291-300 [1996]).
Transvascular delivery of viral particles to tumor cells is hampered by the blood-brain barrier, and particularly the blood-tumor barrier, that results from the interendothelial tight junctions formed by cerebrovascular endothelial cells.
Neuwelt et al. used an intracarotid injection of hypertonic mannitol to osmotically disrupt the blood-brain barrier. They reported that this enhanced the uptake by brain tissue of inactivated HSV-1 particles that were administered immediately afterward by bolus intracarotid injection, but there was no such enhancement when HSV was injected intravenously. (E. A. Neuwelt et al., Delivery of ultraviolet-inactivated 35S-herpesvirus across an osmotically modified blood-brain barrier, J. Neurosurg. 74(3):475-79 [1991]; Also, S. E. Doran et al., Gene expression from recombinant viral vectors in the central nervous system after blood-brain barrier disruption, Neurosurgery 36(5):965-70 [1995]; G. Nilaver et al., Delivery of herpesvirus and adenovirus to nude rat intracerebral tumors after osmotic blood-brain barrier disruption, Proc. Natl. Acad. Sci. USA 92(21):9829-33 [1995]).
Rainov et al. described enhanced transvascular delivery of HSV into gliosarcoma cells in the brains of rats by injecting bradykinin, a vasoactive polypeptide, into the internal carotid artery to disrupt the blood-brain barrier. (N. G. Rainov, Selective uptake of viral and monocrystalline particles delivered intra-arterially to experimental brain neoplasms, Hum. Gene. Ther. 6(12):1543-52 [1995]; N. G. Rainov et al., Long-term survival in a rodent brain tumor model by bradykinin-enhanced intra-arterial delivery of a therapeutic herpes simplex virus vector, Cancer Gene Ther. 5(3):158-62 [1998]). A bradykinin analog, RMP-7, was shown to selectively open the blood-tumor barrier in rats to hrR3 HSV particles without the hypotensive effects associated with the use of bradykinin. (F. H. Barnett et al., Selective delivery of herpes virus vectors to experimental brain tumors using RMP-7, Cancer Gene Ther. 6(1):14-20 [1999]).
There is a definite need for an effective HSV-1-derived vector for cancer therapy, that is virulence impaired and also does not reactivate spontaneously. There is a further definite need for a method of inhibiting malignant cells using such an HSV-1-derived vector that is also capable of reaching tumors of the central nervous system, particularly brain tumors. These and other benefits the present invention provides as described herein.
The present invention is directed to a method of selectively inhibiting the growth of malignant cells in mammals, including humans. The method selectively inhibits the growth of malignant cells of all varieties, and is particularly useful in treating brain tumors and other malignancies of the central nervous system. The method employs an HSV-1-derived viral vector containing a deletion in both copies of the LAT gene and both copies of the ICP34.5 gene of HSV-1, and which is delivered to the malignant cell. One of the advantages of using a combined LAT null and xcex334.5 null HSV-1-derived vector, compared to LAT null mutants or xcex334.5 null mutants alone, is that regardless of the infectious dose, the double mutant does not reactivate from latency. The HSV-1-derived vector of the present invention employed in the method, optionally contains a functional HSV thymidine kinase gene, which can enhance the effectiveness against cancer of drug treatment with gancyclovir or acyclovir.
The combination of LAT null and xcex334.5 null mutations results in a viral vector that does not damage neurons, but grows well and kills most other cells. Alternatively, the method employs an HSV-1-derived vector that contains at least one transcriptional unit of a LAT promoter sequence operatively linked to a nucleic acid having a nucleotide sequence encoding a polypeptide toxic for cells expressing the vector, for example, human interferon-xcex3, interleukin-2, interleukin-4, interleukin-6, interleukin-10, interleukin-12, granulocyte-macrophage colony stimulating factor, tumor necrosis factor-xcex1, Fas ligand, human connexin-43, VP-16, or VP-22.
The present invention relates to a method of expressing a preselected protein in a mammalian cell, employing a vector of the present invention that contains at least one transcriptional unit of a constitutive LAT promoter operatively linked to a gene encoding any preselected protein. When the infected cell expresses, from the LAT promoter, it produces the preselected protein in an elevated amount compared to uninfected homologous cells. Thus, the present invention also relates to a method of treating a genetic defect in a mammal that is based on expressing a desired preselected protein, using a vector in accordance with the present invention, to mitigate or ameliorate a genetic disease condition.
The present invention also relates to a method of detecting a mammalian cell that expresses HSV, employing an HSV-1-derived vector, with the same double deletion of LAT and ICP34.5 genes, and a fluorescent or light-emitting (green fluorescent protein) gene inserted into the vector under the transcriptional control of the LAT promoter. The resulting fluorescence or luminescence allows easy detection of the location of the HSV-1 expressing cell, when tissue sections are examined under a fluorescent microscope or when cells are analyzed with flow-activated cell sorting (FACS).
The present invention is also directed to HSV-1-derived vectors and kits for expressing a preselected protein in a mammalian cell, useful for practicing the method, and to mammalian cells containing the HSV-derived vectors.
These and other advantages and features of the present invention will be described more fully in a detailed description of the preferred embodiments which follows.